Comparison of the effects of ionization mechanism, analyte concentration, and ion “cool-times” on the internal energies of peptide ions produced by electrospray and atmospheric pressure matrix-assisted laser desorption ionization

The propensities of a series of peptide ions produced by both electrospray and atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) to fragment in an ion trap mass spectrometer under various conditions were studied in detail by measuring the extent of fragmentation of precursor ions by collision induced dissociation (CID) as a function of applied resonance excitation RF voltage. For the most basic peptides, the energy required to fragment MH+ ions generated by electrospray exceeded that required to fragment equivalent AP-MALDI ions under identical instrumental conditions; the reverse was observed for a peptide incorporating no basic residues, while peptides of intermediate basicity showed little difference between the ionization methods. This correlation between peptide basicity and the difference in the energy required to induce fragmentation of MH+ ions generated by AP-MALDI and electrospray is attributed primarily to a trend in the internal energies of the ions generated by AP-MALDI (the greater the difference in gas-phase basicities between the matrix and the analyte the greater the internal energy of the analyte ions produced). Furthermore the internal energies of ions produced by AP-MALDI, but not the equivalent ions formed by electrospray, were observed to decrease with decreasing analyte concentration. We attribute this finding to the cooling effect of endothermic dissociation of analyte ion/matrix molecule clusters following the matrix assisted laser desorption step. Time-resolved analyses (measurement of extent of fragmentation of precursor ions by CID as a function of pre-CID "cool times") revealed that cooling periods in excess of 250 ms were required to achieve internal energy equilibrium through cooling collisions with the helium buffer gas. Furthermore, these analyses demonstrated that, even after these extended cooling times, equivalent ions formed by the two ionization techniques showed different propensities to fragment. We conclude that the two different ionization techniques produce ion populations that may differ in their three-dimensional structure.     

Expansion Cooling in the Matrix Plume is Under-Recognized in MALDI Mass Spectrometry

Time-of-flight (TOF) mass spectra for a peptide (Y₆) were obtained by utilizing matrix-assisted infrared laser desorption ionization (IR-MALDI) with glycerol as the matrix and by ultraviolet MALDI with α-cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), and 2,5-dihydroxybenzoic acid (DHB). Collisional activation during ion extraction and exothermicity in the gas-phase proton transfer were found to be unimportant as the driving forces for in-source (ISD) and post-source (PSD) decays, indicating that the thermal energy acquired during photo-ablation is responsible for their occurrence. The temperatures of [Y₆ + H]⁺ in the ‘early’ and ‘late’ matrix plumes were estimated by the kinetic analysis of the ISD and PSD yields, respectively. The order of the temperatures was glycerol < DHB ≈ SA < CHCA in the early plume and glycerol < DHB < SA < CHCA in the late plume. For each matrix, the temperature in the late plume was lower than in the early plume by 300–400 K, which was attributed to expansion cooling. The model (thermalization followed by expansion cooling) proposed to explain the occurrence of both rapid ISD and slow PSD is not only in sharp contrast with but also mutually exclusive with the prevailing explanation that the exothermicity in proton transfer and in-plume collisional activation are the driving forces for ion fragmentation in MALDI. The model also explains why MALDI is more successful for mass spectrometry of labile molecules than other desorption techniques that do not utilize a matrix. Factors affecting the plume temperature are also discussed.     

Do collisions inside the collision cell play a relevant role in CID‐LIFT experiments?

Collision experiments are a valid approach to characterize the ionic species generated by matrix assisted laser desorption ionization (MALDI). When a time-of-flight analyzer is employed, three different approaches are available for these experiments: the postsource decay (PSD), the LIFT and the MALDI-TOF/TOF. The last two are of particular interest because of the overcoming of the PSD problems related to mass calibration of the product ion spectra. Experiments performed by LIFT on linear or cyclic peptides, in presence or in absence of collision gas in the collision cell, gave evidence of an unexpected behavior: the two spectra were practically superimposable, and in the former case only a few new fragmentation channels were activated with low yield. These results mean that the selected ion exhibits a large amount of internal energy, capable of promoting fragmentation processes in the time window corresponding to the flight time between ion source and the acceleration electrode placed after the collision cell. Experiments performed by varying the plume density show that this internal energy uptake occurs in the expanding plume, through multiple collisions. The LIFT data have been compared with those achieved by collisions of ESI-generated [MH](+) ions of angotensin II performed under 'in-source' conditions and by triple-quadrupole experiments. The obtained results show a strong similarity among the spectra, indicating that the internal energy uptake in a MALDI source is comparable with that of 40-eV ions colliding with Ar in a triple-quadrupole instrument.     

Factors that influence the observed fast fragmentation of peptides in matrix-assisted laser desorption

Fragmentation processes that occur very early during matrix-assisted laser desorption ionization (MALDI) of peptides are examined by utilization of delayed pulsed ion extraction with a linear time-of-flight mass spectrometer. The oxidized B chain of bovine insulin (MW=3495. 95 u), which produces a wide range of fragment ions, is utilized as a probe to examine the effects of several experimental parameters on this process. Experimental evidence suggests that this MALDI process is not prompt fragmentation and involves metastable ion decay that is quite different from that which is observed with postsource decay experiments. This conclusion is based upon the significant differences observed in the fragmentation products produced by the two techniques. This metastable ion decay process also appears to be over within the minimum pulse delay period (320 ns) that is possible with the current pulsed ion extraction hardware. These two observations suggest that either different activation processes are involved in the two techniques or that the much different time frame of the methods influences the observed ion decay pathways. This fast MALDI metastable ion fragmentation also is shown to be influenced by both the MALDI matrix and the laser fluence.     

Internal energy transfer in laser desorption/ionization from silicon nanowires

Laser-induced desorption/ionization from silicon nanowires (SiNW) is an emerging method for mass spectrometry of small to medium-size molecules. In this new technique, we examined the internal energy transfer to seven benzylpyridinium thermometer ions and extracted the corresponding internal energy distributions. To explore the effect of the energy-deposition rate on the internal energy transfer, two lasers with significantly different pulse lengths (4 ns vs 22 ps) were utilized as excitation sources. A comparison of ion yields indicated that the SiNW substrates required 5-8 times less laser fluence for ion production than either matrix-assisted laser desorption/ionization (MALDI) or desorption/ionization on silicon (DIOS). In contrast however, the survival yield (SY) values showed that the internal energy transferred to the thermometer ions was more than (ps laser) or comparable to (ns laser) MALDI but it was significantly less than in DIOS. The internal energy transfer was only slightly dependent on laser fluence and on wire density. These effects were rationalized in terms of the confinement of thermal energy in the nanowires and of unimpeded three-dimensional plume expansion. Unlike in MALDI from CHCA and in perfluorophenyl-derivatized DIOS, for desorption from SiNWs the effect of laser pulse length on the internal energy transfer was found to be negligible.     

Fragmentation of poly(amidoamine) dendrimers in matrix-assisted laser desorption

Fragmentation of different generations of poly(amidoamine) dendrimers was explored in five common MALDI matrices: 2,5-dihydroxybenzoic acid (DHB), 4-hydroxy-3-methoxycinnamic acid (FER), α-cyano-4-hydroxycinnamic acid (ACH), 2,4,6-trihydroxyacetophenone (THAP), and 3-hydroxypicolinic acid (HPA). Of these, DHB was the softest matrix and ACH produced significant fragment intensity already at MALDI threshold, FER and THAP being in between. HPA was not a convenient matrix for dendrimers and produced a specific fragmentation pattern. Fragmentation analysis was mainly concentrated on generation G1, which contains already all essential structural elements. Dendrimers showed complicated fragmentation behavior with multiple fragmentation channels in our MALDI experiments. The relative intensities of these channels depended selectively on choice of the matrix and showed dissimilar dependence on the laser pulse energy. This was attributed to different fragmentation mechanisms, due to different protonation pathways, occurring in the same MALDI plume. The fragmentation pathways were proposed for all observed fragmentation channels. All fragmentation sites of protonated ions were found to be directly attached to the protonation sites and the fragmentation was surplus charge driven in this sense. No charge remote fragmentation channels were detected. Cationized dendrimers showed higher stability than the protonated ions.     

Surface modification and laser pulse length effects on internal energy transfer in DIOS

Benzyl-substituted benzylpyridinium (BP) chloride salts were used as a source of thermometer ions to probe the internal energy (IE) transfer in desorption/ionization on porous silicon (DIOS). To modify their wetting properties and the interaction energies with the thermometer ions, the DIOS surfaces were silylated to produce trimethylsilyl- (TMS), amine- (NH2), perfluoroalkyl- (PFA), and perfluorophenyl-derivatized (PFP) surfaces. Two laser sources--a nitrogen laser with pulse length of 4 ns and a mode locked 3 x omega Nd:YAG laser with a pulse length of 22 ps--were utilized to induce desorption/ionization and fragmentation at various laser fluence levels. The corresponding survival yields were determined as indicators of the IE transfer and the IE distributions were extracted. In most cases, with increasing the laser fluence in a broad range (approximately 20 mJ/cm2), no change in IE transfer was observed. For ns excitation, this was in remarkable contrast with MALDI, where increasing the laser fluence resulted in sharply (within approximately 5 mJ/cm2) declining survival yields. Derivatization of the porous silicon surface did not affect the survival yields significantly but had a discernible effect on the threshold fluence for ion production. The IE distributions determined for DIOS and MALDI from alpha-cyano-4-hydroxycinnamic acid reveal that the mean IE value is always lower for the latter. Using the ps laser, the IE distribution is always narrower for DIOS, whereas for ns laser excitation the width depends on surface modification. Most of the differences between MALDI and DIOS described here are compatible with the different dimensionality of the plume expansion and the differences in the activation energy of desorption due to surface modifications.     

Production and fragmentation of multiply charged ions in 'electron-free' matrix-assisted laser desorption/ionization

An unusually large fraction of multiply charged ions is observed in 'electron-free' matrix-assisted laser desorption/ionization (MALDI). Here we investigate how the yield of multiply charged ions depends on experimental parameters in MALDI. It is found to increase if measures are taken to limit the number of electrons in the plume, for example, by using non-metallic MALDI targets or low laser pulse energies. The ionization energy of the matrix is another important parameter that affects the yield of multiply charged ions: matrices with high ionization energies lead to greater intensities of multiply charged ions. It is furthermore proposed that some of the fragment ions observed in MALDI are due to reactions of analyte with electrons in the plume. The possibility of electron capture dissociation of multiply charged ions produced by MALDI is shown.     

The nature of collision-induced dissociation processes of doubly protonated peptides: comparative study for the future use of matrix-assisted laser desorption/ionization on a hybrid quadrupole time-of-flight mass spectrometer in proteomics.

Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.     

Matrix-assisted laser desorption/ionization tandem reflectron time-of-flight mass spectrometry of fullerenes

Atandem reflectron time-of-flight mass spectrometer developed in our laboratory provides a unique opportunity to investigate the collision-induced dissociation of fullerene ions formed by matrix-assisted laser desorption/ionization (MALDI). Specifically, this opportunity arises from the ability to utilize high energy collisional activation (normally available only on tandem sector instruments by using continuous ionization techniques) for ions formed by pulsed laser desorption, whereas most MALDI time-of-flight instruments record product ion mass spectra of ions formed by metastable or postsource decay. In this study we investigate the products of mass-selected and collisionally activated C ₆₀ ⁺ and C ₇₀ ⁺ ions by using different target gases over a range of target gas pressures. In general, heavier target gases produce more extensive fragmentation and improve the mass resolution of lower mass ionic products because a greater portion of these ions are formed by single collisions. Additionally, the tandem time-of-flight instrument utilizes a nonlinear (curved-field) reflectron in the second mass analyzer that enables high energy collision-induced dissociation spectra to be recorded without scanning or stepping the reflectron voltage.     

Ionization in matrix-assisted laser desorption/ionization: singly charged molecular ions are the lucky survivors

A new model for the ionization processes in UV matrix-assisted laser desorption/ionization (MALDI) which accounts for the major phenomena observed is presented and discussed. The model retains elements of earlier approaches, such as photoionization and photochemical reactions, but it redefines these in the light of new working questions, most importantly why only singly charged ions are detected. Based on experimental evidence, the formation of singly and multiply charged clusters by a deficiency/excess of ions and also by photoionization and subsequent photochemical processes is pointed out to be the major ionization processes, which typically occur in parallel. The generation of electrons and their partial loss into the surrounding vacuum and solid, on the one hand, results in a positively charged ion-neutral plume facilitating a high overall ionization yield. On the other hand, these electrons, and also the large excess of protonated matrix ions in the negative ion mode, induce effective ion reneutralization in the plume. These neutralization processes are most effective for the highly charged cluster ions initially formed. Their fragmentation behaviour is evidenced in fast metastable fragmentation characteristics and agrees well with an electron capture dissociation mechanism and the enthalpy transfer upon neutralization forms the rationale for the prominent fragmentation and intense chemical noise accompanying successful MALDI. Within the course of the paper, cross-correlations with other desorption/ionization techniques and with earlier discussions on their mechanisms are drawn.     

Competing ion decomposition channels in matrix-assisted laser desorption ionization

We gauged the internal energy transfer for two dissociative ion decomposition channels in matrix-assisted laser desorption ionization (MALDI) using the benzyltriphenylphosphonium (BTP) thermometer ion [PhCH 2PPh 3] (+). Common MALDI matrixes [alpha-cyano-4-hydroxycinnamic acid (CHCA), 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid, SA), and 2,5-dihydroxycinnamic acid (DHB)] were studied with nitrogen laser (4 ns pulse length) and mode-locked 3 x omega Nd:YAG laser (22 ps pulse length) excitation. Despite the higher fluence required to initiate fragmentation, BTP ions indicated lower internal energy transfer with the picosecond laser in all three matrixes. These differences can be rationalized in terms of phase explosion induced by the nanosecond laser vs a stress-confinement-driven desorption mechanism for the picosecond laser. For the two ion production channels of the BTP thermometer ion, breaking a single bond can result in the formation of benzyl/tropylium ions, F1, or triphenylphosphine ions, F2. In SA and DHB, as well as in CHCA at low fluence levels, the efficiency of these channels (expressed by the branching ratio I F1/ I F2) is moderately in favor of producing tropylium ions, 1 < I F1/ I F2 < 6. As the laser fluence is increased, for CHCA, there is a dramatic shift in favor of the tropylium ion production, with I F1/ I F2 approximately 30 for the nanosecond and the picosecond laser, respectively. This change is correlated with the sudden increase in the BTP internal energies in CHCA in the same laser fluence range. The large changes observed in internal energy deposition for CHCA with laser fluence can account for its ability to induce fragmentation in peptides more readily than SA and DHB.     

Peptide photodissociation at 157 nm in a linear ion trap mass spectrometer

The photodissociation by 157 nm light of singly- and doubly-charged peptide ions containing C- or N-terminal arginine residues was studied in a linear ion trap mass spectrometer. Singly-charged peptides yielded primarily x- and a-type ions, depending on the location of the arginine residue, along with some related side-chain fragments. These results are consistent with our previous work using a tandem time-of-flight (TOF) instrument with a vacuum matrix-assisted laser desorption/ionization (MALDI) source. Thus, the different internal energies of precursor ions in the two experiments seem to have little effect on their photofragmentation. For doubly-charged peptides, the dominant fragments observed in both photodissociation and collisionally induced dissociation (CID) experiments are b- and y-type ions. Preliminary experiments demonstrating fragmentation of multiply-charged ubiquitin ions by 157 nm photodissociation are also presented.     

On the time scale of internal energy relaxation of AP-MALDI and nano-ESI Ions in a quadrupole ion trap

Recently reported results (Konn et al. [14]) on the collisional cooling of atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) and nano-electrospray ionization (nano-ESI) generated ions in a quadrupole ion trap mass spectrometer (QITMS) are inconsistent with measured collisional cooling rates. The work reported here presents a re-examination of those previous results. Collision induced dissociation (CID) has been used to probe various properties of ions contained in a QITMS. It is shown experimentally that when trapping large numbers of ions, an effective dc trapping voltage is induced that varies with changes in the size of the ion cloud. A decrease in the resonant frequency for maximum CID efficiency is observed as the cool time between parent ion isolation and CID is increased. Ion trajectories in a QITMS are simulated to demonstrate how ion density changes over the course of parent ion isolation. The effect of space charge on ion motion is simulated, and Fourier transformations of ion axial motion plus simple calculations corroborate the experimentally observed transient frequency shifts. The relative stability of ions formed by AP-MALDI and nano-ESI is compared under low charge density conditions. These data show that the ions have reached equilibrium internal energy and, thus, that differences in dissociation onsets and “50% fragmentation efficiency points” between the ionization mechanisms are due to the formation of distinct ion conformations as previously shown in reference [28]. The conclusions of Konn et al. [14] are based on invalid experimental procedures as well as inappropriate comparisons of QITMS data to low-pressure FT-ICR data.     

Secondary ion-molecule reactions in matrix-assisted laser desorption/ionization

Ion-molecule charge- and proton-transfer reactions in the desorption plume are considered for the case of matrix-assisted laser desorption/ionization (MALDI) with ultraviolet laser excitation, and it is proposed that they are major determinants of the observed mass spectrum. Specific MALDI phenomena which are discussed include the dominance of singly charged ions and analyte-matrix or analyte-analyte signal suppression. Should any be formed, highly charged products can be reduced by reaction with neutral matrix, yet singly charged ions cannot generally be neutralized in the same manner. Ion suppression effects can also be explained by similar reactions, which in some cases involve interconversion of dissimilar ion types. The plume is proposed often to be more under thermodynamic rather than kinetic control owing to these secondary reactions. UV/MALDI mass spectra should therefore be largely predictable, given sufficient thermodynamic information, and appropriate experimental conditions of sufficient analyte and plume density. Copyright 2000 John Wiley & Sons, Ltd.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of lipids: ionization and prompt fragmentation patterns

Ionization and prompt fragmentation patterns of triacylglycerols, phospholipids (PLs) and galactolipids were investigated using matrix-assisted laser desorption/ionization (MALDI). Positive ions of non-nitrogen-containing lipids appeared only in the sodiated form, while nitrogen-containing lipids were detected as both sodiated and protonated adducts. Lipids containing acidic hydroxyls were detected as multiple sodium adducts or deprotonated ions in the positive and negative modes, respectively, with the exception of phosphatidylcholines. The positive MALDI spectra of triacylglycerols contained prompt fragments equivalent to the loss of RCOO(-) from the neutral molecules. Prompt fragment ions [PL-polar head](+) were observed in the positive MALDI spectra of all phospholipids except phosphatidylcholines. The phosphatidylcholines produced only a minor positive fragment corresponding to the head group itself (m/z 184). Galactolipids did not undergo prompt fragmentation. Post-source decay (PSD) was used to examine the source of prompt fragments. PSD fragment patterns indicated that the lipid prompt fragment ions did not originate from the observed molecular ions (sodiated or protonated), and suggested that the prompt fragmentation followed the formation of highly unstable, probably protonated, precursor ions. Pathways leading to the formation of prompt fragment ions are proposed.     

Matrix-assisted laser desorption/ionization Fourier transform mass spectrometry of luteinizing hormone releasing hormone-metal ion complexes

Complexes of luteinizing hormone releasing hormone (LHRH) with divalent metal ions (Ni, Zn, Cu) have been studied by matrix-assisted laser desorption ionization (MALDI) and Fourier transform mass spectrometry. LHRH-metal complexes were detected in high abundance for all three metals from synthesized samples, particularly in negative ion mode. The mixture of the apopeptide with the metal salts yielded in most cases a very minor signal of metal-complex ions. As opposed to Ni and Zn, copper complex ions were mostly observed as Cu(I) adducts. This can be partly attributed to plume reactions of Cu(I) with the apopeptide. the Cu(II) complexes appeared only for the synthetic complex. We show how to distinguish between the contribution to the overall signal from desorbed complexes and from Cu(I) complexes formed in the MALDI plume.     

Fragmentation of organic ions bearing fixed multiple charges observed in MALDI MS

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI TOF MS) was used to analyze a series of synthetic organic ions bearing fixed multiple charges. Despite the multiple intrinsic charges, only singly charged ions were recorded in each case. In addition to the pseudo‐molecular ions formed by counterion adduction, deprotonation and electron capture, a number of fragment ions were also observed. Charge splitting by fragmentation was found to be a viable route for charge reduction leading to the formation of the observed singly charged fragment ions. Unlike multivalent metal ions, organic ions can rearrange and/or fragment during charge reduction. This fragmentation process will evidently complicate the interpretation of the MALDI MS spectrum. Because MALDI MS is usually considered as a soft ionization technique, the fragment ion peaks can easily be erroneously interpreted as impurities. Therefore, the awareness and understanding of the underlying MALDI‐induced fragmentation pathways is essential for a proper interpretation of the corresponding mass spectra. Due to the fragment ions generated during charge reduction, special care should be taken in the MALDI MS analysis of multiply charged ions. In this work, the possible mechanisms by which the organic ions bearing fixed multiple charges fragment are investigated. With an improved understanding of the fragmentation mechanisms, MALDI TOF MS should still be a useful technique for the characterization of organic ions with fixed multiple charges.     

Internal energies of analyte ions generated from different matrix-assisted laser desorption/ionization matrices

A transfer of energy into the internal modes of the matrix and analyte is expected to occur during matrix-assisted laser desorption/ioniziation (MALDI) processes. Both the physical and thermochemical properties of the MALDI matrix used can influence the ion internal energy and analyte ion fragmentation. Here we report the effect of several MALDI matrices on the relative internal energy of the 2'-deoxyadenylyl-(3',5')-2'-deoxyguanosine (AG) anion. Relative internal energies were probed by low-energy collision-induced dissociation in a Fourier transform ion cyclotron resonance mass spectrometer. Sublimation temperatures of the matrices under study were also determined and found to lie between 409 and 455 K. Analyte ion internal and initial kinetic energies did not correlate with matrix sublimation temperatures. In contrast, a strong correlation between the relative internal energy of the analyte anions and the gas-phase basicity of the matrix anions was found. These results suggest that gas-phase proton transfer reactions play an important role in MALDI analyte ion formation and influence their internal energy and fragmentation behavior. Copyright 2000 John Wiley & Sons, Ltd.     

Matrix suppression and analyte suppression effects of quaternary ammonium salts in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: an investigation of suppression mechanism

In the matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI TOF MS) analysis of some quaternary ammonium salts (QASs), very clean spectra of the quaternary ammonium ions were recorded with a strong matrix suppression effect (MSE). The QASs also showed a considerable analyte suppression effect (ASE). It was demonstrated that the MSE and ASE of the QASs can be explained well by the cluster ionization model. According to this model, MALDI ions are formed from charged matrix/analyte clusters. Various analyte ions and matrix ions might coexist in the cluster, and they will compete for the limited number of net charges available. If enough quaternary ammonium ions are present in the cluster, they will take away the net charges, thus resulting in the MSE and ASE. Our results also suggest that ‘the cluster ionization model’ is not in conflict with ‘the theory of ionization via secondary gas‐phase reactions’. The initial MALDI ions produced from charged matrix/analyte clusters will collide with other molecules or ions in the MALDI plume. Depending on the properties of the initial ions and the composition of the MALDI plume, secondary gas‐phase reactions might result from these collisions. The final ions observed are the combined results of ‘cluster ionization’ and ‘ionization via secondary gas‐phase reactions’. Copyright © 2009 John Wiley & Sons, Ltd.